Chronic morphine use has been associated with an increased incidence of many diseases, and animal studies have demonstrated that morphine can alter a number of immune parameters. Although these effects of morphine are very well established, it still remains unresolved the receptor type involved in morphine mediated effects on immune parameters. In addition it is still controversial if it is the central mu-opioid receptors or receptors present on target cells that mediates morphine's immunosupressive effects. Gene targeting methodology makes it possible to eliminate or knock-out the mu opioid receptor in mice (MORKO), providing a powerful too] to address this question. In such animals, both direct and indirect effects of morphine on the immune system can no longer be mediated by mu opioid receptors, making it possible to evaluate the role of other classical opioid receptors, including delta and kappa types as well as non-classical naloxone-insensitive receptors in morphine mediated immune functions. The objective of this proposal is to determine the contribution of these receptors in modulating the immune system and to gain insight into the intracellular mechanisms by which morphine mediates its effects. We propose to determine the role of Mu-opioid receptors in morphine mediated immunosuppression by using wild type and MORKO mice. These studies will include the role of Mu receptors in the brain on activation of hypothalamic-pituitary-adrenal axis. Subsequently, we will investigate the relative role of peripheral Mu opioid receptors in morphine mediated immunosuppression. The mechanism by which Mu-opioid receptors modulate cytokine gene expression will also be studied. Finally, cDNA libraries constructed from the immune cells of MORKO mice will be used to clone and characterize the naloxone-insensitive morphine binding site. Investigations into the mechanism of morphine action on immune cells would help us understand how drug abuse increases susceptibility to HIV infection. Experiments outlined in this proposal address some of the fundamentals of morphine action on immune cells and these studies will help in the development of biotherapies to prevent immunosuppression while retaining analgesic properties of morphine.